How do you measure the concentration of purified GFP?

How do you measure the concentration of purified GFP?

• A. By using a colorimeter to assess absorbance at 600 nm
• B. Using a spectrophotometer at a wavelength of 488 nm
• C. By measuring fluorescence intensity through a microscope
• D. Using liquid chromatography-mass spectrometry

Answer: (B)

The measurement of purified GFP concentration is best performed using a spectrophotometer at a wavelength of 488 nm because this wavelength corresponds to the peak absorbance for GFP. When GFP is excited by light at this specific wavelength, it absorbs significant amounts of light, allowing for an accurate quantification of its concentration based on the Beer-Lambert law. This law indicates that the absorbance of light by a solution is directly proportional to the concentration of the absorbing species in the solution. Using a colorimeter at 600 nm would not be appropriate, as GFP does not have a significant absorbance peak in that region of the spectrum, leading to inaccurate concentration measurements. Measuring fluorescence intensity through a microscope can provide information about the GFP's activity and localization, but it does not give a direct concentration value. Liquid chromatography-mass spectrometry is a highly accurate method for analyzing proteins and metabolites, but it is typically more complex and not specifically designed for straightforward concentration measurements of proteins like GFP in simple solutions, especially in a laboratory setting focused on absorbance readings. Hence, the most reliable and direct method for assessing GFP concentration is through a spectrophotometer at 488 nm.

The measurement of purified GFP concentration is best performed using a spectrophotometer at a wavelength of 488 nm because this wavelength corresponds to the peak absorbance for GFP. When GFP is excited by light at this specific wavelength, it absorbs significant amounts of light, allowing for an accurate quantification of its concentration based on the Beer-Lambert law. This law indicates that the absorbance of light by a solution is directly proportional to the concentration of the absorbing species in the solution.

Using a colorimeter at 600 nm would not be appropriate, as GFP does not have a significant absorbance peak in that region of the spectrum, leading to inaccurate concentration measurements. Measuring fluorescence intensity through a microscope can provide information about the GFP's activity and localization, but it does not give a direct concentration value. Liquid chromatography-mass spectrometry is a highly accurate method for analyzing proteins and metabolites, but it is typically more complex and not specifically designed for straightforward concentration measurements of proteins like GFP in simple solutions, especially in a laboratory setting focused on absorbance readings. Hence, the most reliable and direct method for assessing GFP concentration is through a spectrophotometer at 488 nm.